CN112695024A - Linoleic acid isomerase and application thereof in conjugated linoleic acid production - Google Patents
Linoleic acid isomerase and application thereof in conjugated linoleic acid production Download PDFInfo
- Publication number
- CN112695024A CN112695024A CN201911011728.3A CN201911011728A CN112695024A CN 112695024 A CN112695024 A CN 112695024A CN 201911011728 A CN201911011728 A CN 201911011728A CN 112695024 A CN112695024 A CN 112695024A
- Authority
- CN
- China
- Prior art keywords
- linoleic acid
- ala
- conjugated linoleic
- isomerase
- leu
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- OYHQOLUKZRVURQ-IXWMQOLASA-N linoleic acid Natural products CCCCC\C=C/C\C=C\CCCCCCCC(O)=O OYHQOLUKZRVURQ-IXWMQOLASA-N 0.000 title claims abstract description 152
- JBYXPOFIGCOSSB-GOJKSUSPSA-N 9-cis,11-trans-octadecadienoic acid Chemical compound CCCCCC\C=C\C=C/CCCCCCCC(O)=O JBYXPOFIGCOSSB-GOJKSUSPSA-N 0.000 title claims abstract description 110
- 229940108924 conjugated linoleic acid Drugs 0.000 title claims abstract description 83
- 235000020778 linoleic acid Nutrition 0.000 title claims abstract description 59
- 108090000769 Isomerases Proteins 0.000 title claims abstract description 52
- 102000004195 Isomerases Human genes 0.000 title claims abstract description 52
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 15
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- OYHQOLUKZRVURQ-HZJYTTRNSA-N Linoleic acid Chemical compound CCCCC\C=C/C\C=C/CCCCCCCC(O)=O OYHQOLUKZRVURQ-HZJYTTRNSA-N 0.000 claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 10
- 238000000034 method Methods 0.000 claims description 19
- 150000001413 amino acids Chemical class 0.000 claims description 16
- 238000012258 culturing Methods 0.000 claims description 13
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Abstract
The invention discloses linoleic acid isomerase and application thereof in conjugated linoleic acid production, and belongs to the technical field of protein engineering and microbial engineering. The linoleic acid isomerase derived from the bifidobacterium can be used for producing conjugated linoleic acid, the recombinant escherichia coli containing the linoleic acid isomerase is added into a reaction system containing linoleic acid for reaction for 3 hours, so that the conversion rate of the conjugated linoleic acid can reach 12.1-42.1%, the content of cis9 and trans11-CLA in the conjugated linoleic acid can reach 84.3-89.1%, the result is that the safety is further high, the yield is high, most of the produced conjugated linoleic acid monomer is cis9 through a genetic engineering means, and the microorganism of trans11-CLA provides full theoretical support.
Description
Technical Field
The invention relates to linoleic acid isomerase and application thereof in conjugated linoleic acid production, and belongs to the technical field of protein engineering and microbial engineering.
Background
Conjugated Linoleic Acid (CLA) is a generic term for a series of fatty acids containing Conjugated double bonds, with a variety of positional and geometric isomers. Researches show that conjugated linoleic acid has physiological effects of resisting cancer, reducing blood fat, resisting atherosclerosis, regulating energy metabolism, enhancing immunity of organisms, promoting growth and development and the like, and is widely applied to the fields of medicines, foods, cosmetics and the like, and cis9, trans11-CLA and trans10 and cis12-CLA are two isomers with the most physiological activity in conjugated linoleic acid isomers. Thus, there is a great demand in the market for cis9, trans11-CLA and trans10, cis 12-CLA.
Natural conjugated linoleic acid is mainly present in rumen animals, certain plants and marine organisms, and the natural conjugated linoleic acid is mainly present in the forms of cis9, trans11-CLA, and has extremely high physiological activity, but the content of the natural conjugated linoleic acid is extremely low, so that the demand of the market for the conjugated linoleic acid is difficult to meet. Therefore, methods for artificially synthesizing conjugated linoleic acid have been gradually developed.
At present, the methods for artificially synthesizing conjugated linoleic acid mainly include chemical synthesis methods and microbial synthesis methods. The chemical synthesis method can cause generation of a plurality of toxic byproducts, and has toxic effects on the environment and human body, and the conjugated linoleic acid isomers prepared by the chemical synthesis method are various and are difficult to be effectively separated, so that the chemical synthesis method cannot really realize large-scale industrial production of the conjugated linoleic acid. Compared with a chemical synthesis method, the microbial synthesis method has the advantages of less pollution and single type of the obtained conjugated linoleic acid isomer, so the microbial synthesis method is a method which has great potential and can realize large-scale industrial production of the conjugated linoleic acid.
However, the existing microbiological synthesis method still has the following defects:
firstly, most of microorganisms capable of producing conjugated linoleic acid with high yield are pathogenic bacteria, have great safety problem, and can not be directly used as conjugated linoleic acid production strains for industrial production, such as vibrio fibrisolvens butyrate, propionibacterium, clostridium sporogenes and the like;
secondly, the yield of conjugated linoleic acid produced by part of microorganisms capable of producing conjugated linoleic acid with high yield is not high, and the production efficiency is too low if the microorganisms are used as conjugated linoleic acid production strains for industrial production, such as lactobacillus plantarum ZS2058 (particularly, the reference documents: Qihui, populus, and the like; the research on the mechanism of bioconversion of conjugated linoleic acid by lactobacillus plantarum ZS2058 [ D ], Jiangnan university, 2017),
the above defects all make the existing microbial synthesis method unable to really realize large-scale industrial production of conjugated linoleic acid, so it is urgently needed to find a conjugated linoleic acid production strain with high safety and high yield to overcome the above defects.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to provide a linoleic acid isomerase which can be used for producing conjugated linoleic acid.
[ solution ]
In order to solve the above problems, the present invention provides a Linoleic acid isomerase (EC 5.2.1.5), wherein the Linoleic acid isomerase is:
(a) a protein consisting of an amino acid sequence shown by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4; or,
(b) and (b) the protein which is derived from the protein (a) and has linoleate isomerase activity, wherein the amino acid sequence in the protein (a) is subjected to substitution, deletion or addition of one or more amino acids.
In one embodiment of the present invention, the linoleate isomerase consisting of the amino acid sequence shown in SEQ ID No.1 is derived from Bifidobacterium breve (Bifidobacterium breve), the linoleate isomerase consisting of the amino acid sequence shown in SEQ ID No.2 is derived from Bifidobacterium longum (Bifidobacterium longum), the linoleate isomerase consisting of the amino acid sequence shown in SEQ ID No.3 is derived from Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum), and the linoleate isomerase consisting of the amino acid sequence shown in SEQ ID No.4 is derived from Bifidobacterium odonta (Bifidobacterium dentium).
The invention also provides a gene, and the gene codes the linoleic acid isomerase.
In one embodiment of the invention, the nucleotide sequence of the gene is shown as SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.
The invention also provides a recombinant plasmid which carries the gene.
In one embodiment of the present invention, the vector of the recombinant plasmid is a pET-28a (+) vector.
The invention also provides a host cell, which carries the gene or the recombinant plasmid.
In one embodiment of the invention, the host cell is E.coli.
The invention also provides the application of the linoleic acid isomerase, the gene, the recombinant plasmid or the host cell in the production of conjugated linoleic acid.
In one embodiment of the invention, the conjugated linoleic acid is cis9, trans11-CLA and/or trans9, trans 11-CLA.
The invention also provides a method for producing the conjugated linoleic acid, which comprises the steps of inoculating the host cell into a culture medium, and culturing the host cell to OD under the conditions that the temperature is 35-40 ℃ and the rotating speed is 150-250 rpm6000.4-0.6 to obtain a culture solution A; adding IPTG with the final concentration of 0.01-1.0 mM into the culture solution A, and carrying out induced culture for 12-16 h under the conditions that the temperature is 15-20 ℃ and the rotating speed is 150-250 rpm to obtain a culture solution B; will be provided withCentrifuging the culture solution B, and collecting wet thalli; adding wet bacteria into a reaction system containing linoleic acid by taking the linoleic acid as a substrate, and reacting at the temperature of 35-40 ℃ and the rotating speed of 150-250 rpm to obtain a reaction solution rich in conjugated linoleic acid; extracting the reaction liquid rich in the conjugated linoleic acid to obtain the conjugated linoleic acid.
In one embodiment of the present invention, the method comprises inoculating the above host cells into a culture medium, and culturing the cells at 37 ℃ and 200rpm to OD6000.4-0.6 to obtain a culture solution A; adding IPTG with the final concentration of 0.01-1.0 mM into the culture solution A, and carrying out induced culture for 12-16 h under the conditions that the temperature is 18 ℃ and the rotating speed is 200rpm to obtain a culture solution B; centrifuging the culture solution B, and collecting wet thalli; adding wet thalli into a reaction system containing linoleic acid by taking the linoleic acid as a substrate, and reacting at the temperature of 37 ℃ and the rotating speed of 200rpm to obtain a reaction solution rich in conjugated linoleic acid; extracting the reaction liquid rich in the conjugated linoleic acid to obtain the conjugated linoleic acid.
In one embodiment of the invention, the conjugated linoleic acid is cis9, trans11-CLA and/or trans9, trans 11-CLA.
In one embodiment of the present invention, the reaction system comprises a buffer and linoleic acid.
In one embodiment of the present invention, the pH of the buffer is 6 to 7.
In one embodiment of the invention, the buffer has a pH of 6.5.
In one embodiment of the invention, the buffer is potassium phosphate buffer.
In one embodiment of the invention, the concentration of the linoleic acid in the reaction system is 0.05-0.15 mg/mL.
In one embodiment of the present invention, the concentration of linoleic acid in the reaction system is 0.1 mg/mL.
In one embodiment of the present invention, the concentration of the wet cells in the reaction system is 0.5 to 2 mg/mL.
In one embodiment of the present invention, the concentration of the wet cells in the reaction system is1 mg/mL.
In one embodiment of the invention, the medium is LB medium.
The invention also provides a method for producing the linoleic acid isomerase, which comprises the steps of adding the host cells into a culture medium, culturing at the temperature of 35-40 ℃ and the rotating speed of 150-250 rpm to obtain a culture solution rich in linoleic acid isomerase, and extracting the culture solution rich in linoleic acid isomerase to obtain the linoleic acid isomerase.
In an embodiment of the present invention, the method comprises adding the above host cells into a culture medium, culturing at 37 ℃ and 200rpm to obtain host cells rich in linoleic acid isomerase, and extracting the host cells rich in linoleic acid isomerase to obtain linoleic acid isomerase.
In one embodiment of the invention, the medium is LB medium.
[ advantageous effects ]
(1) The linoleic acid isomerase derived from Bifidobacterium breve and having an amino acid sequence shown in SEQ ID No.1 can be used for producing conjugated linoleic acid, and the recombinant Escherichia coli containing the linoleic acid isomerase can be added into a reaction system containing linoleic acid for reaction for 3 hours, so that the conversion rate of the conjugated linoleic acid can be up to 42.1%, and the cis9 and trans11-CLA contents in the conjugated linoleic acid can be up to 89.1%.
(2) The linoleic acid isomerase derived from Bifidobacterium longum (Bifidobacterium longum) and having an amino acid sequence shown in SEQ ID No.2 can be used for producing conjugated linoleic acid, and the recombinant Escherichia coli containing the linoleic acid isomerase can be added into a reaction system containing linoleic acid for reaction for 3 hours, so that the conversion rate of the conjugated linoleic acid can reach 12.1 percent, and the content of cis9 and trans11-CLA in the conjugated linoleic acid can reach 84.3 percent.
(3) The linoleic acid isomerase derived from Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) with an amino acid sequence shown as SEQ ID No.3 can be used for producing conjugated linoleic acid, recombinant escherichia coli containing the linoleic acid isomerase can be added into a reaction system containing linoleic acid for reaction for 3 hours, so that the conversion rate of the conjugated linoleic acid can reach 19.5%, the content of cis9 and trans11-CLA in the conjugated linoleic acid can reach 88.9%, and the result shows that the safety is high, the yield is high, the produced conjugated linoleic acid monomer is mostly cis9 by means of genetic engineering, and the microorganisms of the trans11-CLA provide full theoretical support.
(4) The linoleic acid isomerase derived from Bifidobacterium dentis (SEQ ID No. 4) and having an amino acid sequence shown in SEQ ID No.4 can be used for producing conjugated linoleic acid, and the recombinant Escherichia coli containing the linoleic acid isomerase can be added into a reaction system containing linoleic acid for reaction for 3 hours, so that the conversion rate of the conjugated linoleic acid can reach 13.5 percent, and the content of cis9 and trans11-CLA in the conjugated linoleic acid can reach 87.1 percent.
Drawings
FIG. 1: effect of IPTG concentration on the conversion of conjugated linoleic acid of recombinant E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21(DE3)/pET28 a-bdi.
FIG. 2: the conjugated linoleic acid isomer type and the ratio of each conjugated linoleic acid isomer in the conjugated linoleic acid produced by the recombinant Escherichia coli E.coli BL21(DE3)/pET28 a-bbi.
FIG. 3: coli BL21(DE3)/pET28a-bli, and the conjugated linoleic acid isomer type and the ratio of the conjugated linoleic acid isomers in the conjugated linoleic acid produced by the recombinant Escherichia coli E.
FIG. 4: coli BL21(DE3)/pET28a-bpi, and the conjugated linoleic acid isomer type and the ratio of each conjugated linoleic acid isomer.
FIG. 5: the conjugated linoleic acid isomer type and the ratio of each conjugated linoleic acid isomer in the conjugated linoleic acid produced by the recombinant Escherichia coli E.coli BL21(DE3)/pET28 a-bdi.
Detailed Description
The invention will be further illustrated with reference to specific examples.
Coli (Escherichia coli) DH5 α, e.coli BL21(DE3) referred to in the examples below were purchased from general biotechnology limited; the pET-28a (+) vector referred to in the examples below was purchased from Invitrogen; the bacterial genome DNA extraction kit and the plasmid mini-extraction kit related in the following examples were purchased from Tiangen Biochemical technology (Beijing) Ltd, and the models thereof are DP302 and DP103, respectively.
The media involved in the following examples are as follows:
MRS solid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·H2O0.05 g/L, Tween 801mL/L, agar 15g/L, cysteine hydrochloride 0.5 g/L.
MRS liquid medium: 10g/L of peptone, 10g/L of beef extract, 20g/L of glucose, 2g/L of sodium acetate, 5g/L of yeast powder and 2g/L, K of diammonium hydrogen citrate2HPO4·3H2O 2.6g/L、MgSO4·7H2O 0.1g/L、MnSO4·H2O0.05 g/L, Tween 801mL/L and cysteine hydrochloride 0.5 g/L.
LB liquid medium: 10g/L tryptone, 5g/L yeast extract and 10g/L sodium chloride, 100. mu.g/mL kanamycin was added before use.
LB solid medium: 10g/L tryptone, 5g/L yeast extract, 10g/L sodium chloride and 15g/L agar, and 100. mu.g/mL kanamycin was added before use.
The detection methods referred to in the following examples are as follows:
the detection method of the specific enzyme activity of the linoleic acid isomerase comprises the following steps: collecting thallus, adding the thallus into KPB buffer solution (pH 6.5), and crushing the thallus with glass beads to obtain cell crushing solution; centrifuging the cell disruption solution at 8000g for 10min, and collecting supernatant to obtain crude enzyme solution; adjusting the protein content in the crude enzyme solution to be 0.5mg/mL, and subpackaging the adjusted crude enzyme solution into 6 reaction glass bottles, wherein each glass bottle contains 1 mL; respectively adding linoleic acid with the final concentration of 0.1mg/mL into a glass bottle, and reacting at 37 ℃ for 60min to obtain reaction liquid; after the reaction is finished, quickly adding isopropanol and n-hexane into the reaction solution, extracting fatty acid, and determining the content change of the fatty acid (the detection method of the content change of the fatty acid refers to the following detection methods of the conversion rate of conjugated linoleic acid, the type of conjugated linoleic acid isomer in the conjugated linoleic acid and the ratio of each conjugated linoleic acid isomer in the conjugated linoleic acid), so as to calculate the specific enzyme activity; the specific enzyme activity (U/mg) ═ W/(T multiplied by M), wherein W is the mass (mu g) of the conjugated linoleic acid generated by the reaction, T is the reaction time (min), and M is the mass (mg) of the sample to be tested
Wherein, the specific enzyme activity of the linoleic acid isomerase is defined as: the amount of enzyme required to convert 1mg conjugated linoleic acid to yield 1mg conjugated linoleic acid in U/mg at 37 ℃ and pH 6.5 in 1 min.
The method for detecting the conversion rate of the conjugated linoleic acid, the type of the conjugated linoleic acid isomer in the conjugated linoleic acid and the ratio of each conjugated linoleic acid isomer in the conjugated linoleic acid comprises the following steps: adding isopropanol and n-hexane into the reaction solution according to the proportion of 1mL of reaction solution, 1mL of isopropanol and 2mL of n-hexane to obtain a mixed solution; carrying out vortex oscillation on the mixed solution for 30 s; standing and layering; transferring the n-hexane layer on the upper layer into a clean spiral glass bottle, and blowing nitrogen to dry; then 400 μ L of methanol was added and vortexed for 30 s; adding 40 mu L of diazomethane into each glass bottle, carrying out methyl esterification, reacting for 15min, and if the color is not faded, representing that the methyl esterification is more sufficient; blowing nitrogen to dry the fully methyl-esterified liquid, respectively adding 200 mu L of n-hexane for redissolving, centrifuging, transferring the supernatant into a chromatographic sampling bottle, and temporarily storing the supernatant until GC-MS detection;
wherein the conversion rate of conjugated linoleic acid (mass of conjugated linoleic acid/mass of linoleic acid in control group) × 100%.
Example 1: screening of Gene encoding linoleate isomerase
The method comprises the following specific steps:
bifidobacterium breve CGMCC No.11828 (described in the patent application text with the publication number of CN 105925514A) is used for collecting transcriptomic data under the stress of linoleic acid by a PacBio sequencing platform, and the sampling time points are 3h, 8h and 15h respectively. It was found through the biological information analysis that the gene transcription level of Bifidobacterium breve (Bifidobacterium breve) CGMCC No.11828 was increased by 8 genes at three time points, and the 8 genes were annotated as genes encoding "unknown protein 1", "melibiose carrier protein", "ribokinase", linoleic acid hydratase "," unknown protein 2 "," transcription regulatory protein "," ribose-bound ABC channel protein 1 "and" ribose-bound ABC channel protein 2 "according to the variation of the transformation level, wherein the gene encoding" unknown protein 1 "was 68-fold increased in 8 hours as compared with 3 hours, and the gene encoding" unknown protein 1 "was 3.5-fold and 8.2-fold as compared with 3 hours, and it did not form gene clusters with other genes, and therefore, it was presumed that the possibility of the gene participating in CLA transformation was high (" the amino acid sequence of unknown protein 1 "is shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.1, and No.2, The nucleotide sequence of the gene for coding the unknown protein 1 is shown as SEQ ID No. 5).
Other genes possibly involved in CLA transformation were obtained from Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) and Bifidobacterium odonta (Bifidobacterium dendum) by the same method (the genes possibly involved in CLA transformation obtained from Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) and Bifidobacterium odonta (Bifidobacterium dendum), respectively, were annotated as genes encoding "unknown protein 3", "unknown protein 4" and "unknown protein 5", respectively, wherein the amino acid sequence of "unknown protein 3" is as shown in SEQ ID No.2, the nucleotide sequence of the gene encoding "unknown protein 3" is as shown in SEQ ID No.6, the amino acid sequence of "unknown protein 4" is as shown in SEQ ID No.3, the nucleotide sequence of the gene encoding "unknown protein 4" is as shown in SEQ ID No.7, and the amino acid sequence of "5" is as shown in SEQ ID No.4, The nucleotide sequence of the gene for coding the unknown protein 5 is shown as SEQ ID No. 8).
Example 2: cloning of Gene encoding linoleate isomerase
The method comprises the following specific steps:
selecting a bacterial liquid of Bifidobacterium breve (CGMCC No. 11828) from a bacteria-retaining tube, streaking the bacterial liquid on an MRS solid culture medium, and culturing the bacterial liquid in a constant-temperature anaerobic workstation at 37 ℃ for 48h to obtain a single bacterial colony; selecting a single colony to inoculate in an MRS liquid culture medium, continuously standing and culturing for 24h in a constant-temperature anaerobic workstation at 37 ℃, and continuously activating for 3 generations to obtain activated bacterial liquid; inoculating the activated bacterial liquid into an MRS liquid culture medium according to the inoculation amount of 1% (v/v), and culturing for 24 hours in a constant-temperature anaerobic workstation at 37 ℃ to obtain bacterial suspension; centrifuging the obtained bacterial suspension for 10min at 25 ℃ and 12000g to obtain wet thalli; extracting genome DNA in wet thalli by using a bacterial genome DNA extraction kit, and amplifying bbi through a PCR reaction; after the PCR reaction is finished, obtaining an amplification product, purifying the amplification product, and verifying the band size of the amplification product through 1% agarose gel electrophoresis to obtain bbi (the bbi gene is the gene encoding the unknown protein 1); wherein, the primers used for the amplification bbi are shown in Table 1;
the PCR reaction system comprises: KOD 1. mu. L, ddH2O 29. mu.L, 1. mu.L of each of the upstream and downstream primers, 1. mu. L, dNTP 5. mu.L of genomic DNA, 10 × reactionbuffer 5. mu.L, and Mg2+3μL；
The PCR reaction conditions are as follows: 95 ℃ for 5 min; circulating for 30 times (95 deg.C, 30 s; 55 deg.C, 30 s; 68 deg.C, 1 min); at 68 ℃ for 5 min; 12 ℃ for 5 min.
Obtaining bli (the bli gene is a gene encoding "unknown protein 3"), bpi (the bpi gene is a gene encoding "unknown protein 4") and bdi (the bdi gene is a gene encoding "unknown protein 5") from Bifidobacterium longum (Bifidobacterium longum), Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum) and Bifidobacterium odonta (Bifidobacterium denatum) by the same method as that of bbi; the primers used for amplification of bli, bpi and bdi are shown in Table 1.
TABLE 1 primer sequences
Example 3: expression of linoleate isomerase in colibacillus
The method comprises the following specific steps:
introducing the pET-28a (+) vector into E.coli DH5 alpha to obtain E.coli DH5 alpha/pET 28 a; coli DH5 alpha/pET 28a was streaked on LB solid medium (containing 10. mu.g/mL kanamycin), and cultured in a 37 ℃ incubator for 18 hours to obtain a single colony; selecting a single colony, inoculating the single colony in an LB liquid culture medium (containing 10 mu g/mL kanamycin), culturing for 14h in a shaker at 37 ℃ and 200rpm, and continuously activating for 3 generations to obtain activated bacterial liquid; inoculating the activated bacterial liquid into an LB liquid culture medium (containing 10 mu g/mL kanamycin) according to the inoculation amount of 1% (v/v), and culturing for 14h in a shaker at 37 ℃ and 200rpm to obtain bacterial suspension; centrifuging the obtained bacterial suspension for 10min at 25 ℃ and 12000g to obtain wet thalli; extracting a pET-28a (+) vector in the wet thalli by using a plasmid miniextraction kit; the obtained pET-28a (+) vector was applied with 50. mu.L of ddH2And O is redissolved and stored at-20 ℃.
The obtained pET-28a (+) vector and the bbi, bli, bpi, bdi genes obtained in example 2 were digested with restriction enzymes Hind III and Nde I, and then T was used4The ligase ligates the digested and purified DNAs to obtain ligation products, wherein the specific ligation system is shown in Table 2.
After the obtained ligation products were ligated overnight at 16 ℃ for 15h, they were transformed into E.coli DH5 alpha competent cells; the transformed E.coli DH5 alpha competent cells were spread on LB solid medium (containing 10. mu.g/mL kanamycin) and cultured by inversion at 37 ℃ for 24 hours; and (3) selecting positive transformants, extracting plasmids, and obtaining recombinant plasmids pET28a-bbi, pET28a-bli, pET28a-bpi and pET28a-bdi as a result of sequencing verification showing that the connection is successful.
The obtained recombinant plasmids pET28a-bbi, pET28a-bli, pET28a-bpi and pET28a-bdi were introduced into E.coli BL21(DE3) respectively to obtain recombinant E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21(DE3)/pET28 a-bdi.
The obtained recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21(DE3)/pET28a-bdi are respectively streaked on an LB solid culture medium and cultured in a constant temperature incubator at 37 ℃ for 18h to obtain single colonies; selecting single colonies, respectively inoculating the single colonies in an LB liquid culture medium, culturing for 14h in a shaking table at 37 ℃ and 200rpm, and continuously activating for 3 generations to obtain activated bacterial liquid; respectively inoculating the activated bacterial liquids into LB liquid culture medium according to the inoculation amount of 1% (v/v), and culturing for 12h under the conditions that the temperature is 37 ℃ and the rotating speed is 200rpm to obtain fermentation liquid; centrifuging the fermentation liquor at 4 deg.C and 12000g for 10min to obtain wet thallus; crushing the wet thallus, and centrifuging for 10min at 4 ℃ and 12000g to obtain a cell crushing supernatant; detecting the activity of linoleic acid isomerase in the obtained cell disruption supernatant, wherein the detection result is as follows:
the activity of linoleic acid isomerase in the cell disruption supernatant obtained by the fermentation of recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bbi is 6.7U/mg, the activity of linoleic acid isomerase in the cell disruption supernatant obtained by the fermentation of recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bli is 1.7U/mg, the activity of linoleic acid isomerase in the cell disruption supernatant obtained by the fermentation of recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bpi is 1.8U/mg, and the activity of linoleic acid isomerase in the cell disruption supernatant obtained by the fermentation of recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bdi is 1.4U/mg. As can be seen, the recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21(DE3)/pET28a-bdi can successfully express linoleate isomerase.
TABLE 2 connection System
Example 4: application of recombinant escherichia coli
The method comprises the following specific steps:
the activated bacterial liquids of the recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21(DE3)/pET28a-bdi obtained in example 3 were inoculated into LB liquid medium in an inoculation amount of 1% (v/v), and cultured at 37 ℃ and 200rpm until OD is reached600After 0.4 to 0.6, IPTG was added to the medium to a final concentration of 0mM, 0.05mM, 0.1mM, 0.3mM, 0.5mM, 0.8mM, 1.0mM, respectively, and the medium was further subjected to induction culture at 18 ℃ and 200rpm for 15 hours to obtain a culture solution; centrifuging the culture solution at 25 deg.C and 12000g for 10min to obtain wet thallus; the wet thalli is respectively suspended back to KPB buffer solution (pH is 6.5) according to the concentration of 0.5mg wet thalli/mL, then linoleic acid with final concentration of 0.01mg/mL, 0.05mg/mL, 0.1mg/mL and 0.5mg/mL is respectively added into the KPB buffer solution, and the mixture is reacted for 3 hours under the conditions of 37 ℃ and 200 rpm; after the reaction is finished, the conversion rate of the conjugated linoleic acid in the reaction liquid is detected, the type of conjugated linoleic acid isomers in the obtained conjugated linoleic acid and the ratio of each conjugated linoleic acid isomer are detected, and the detection results are shown in figures 1-5.
As shown in FIG. 1, when the final concentration of IPTG was 0.1mM, the conversion rate of conjugated linoleic acid was the highest in the reaction solutions obtained by the reactions of recombinant E.coli BL21(DE3)/pET28a-bbi, E.coli BL21(DE3)/pET28a-bli, E.coli BL21(DE3)/pET28a-bpi and E.coli BL21(DE3)/pET28 a-bdi.
As shown in FIGS. 2 to 5, when the final concentration of IPTG is 0.1mM, the conversion rate of conjugated linoleic acid in the reaction solution obtained by the reaction of recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bbi can reach 42.1%, wherein 89.1% is cis9, trans11-CLA, 1% is trans10, cis12-CLA, 9.9% is trans9, and trans 11-CLA;
when the final concentration of IPTG is 0.1mM, the conversion rate of conjugated linoleic acid in a reaction liquid obtained by the reaction of recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bli is 12.1 percent, wherein 84.3 percent is cis9, trans11-CLA, 1.2 percent is trans10, cis12-CLA, 4.5 percent is trans9 and trans 11-CLA;
when the final concentration of IPTG is 0.1mM, the conversion rate of conjugated linoleic acid in a reaction solution obtained by the reaction of recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bpi is 19.5 percent, wherein 88.9 percent is cis9, trans11-CLA, 0.98 percent is trans10, cis12-CLA, 10.1 percent is trans9 and trans 11-CLA;
when the final concentration of IPTG is 0.1mM, the conversion rate of conjugated linoleic acid in a reaction liquid obtained by the reaction of recombinant Escherichia coli E.coli BL21(DE3)/pET28a-bdi is 13.5%, wherein 87.1% is cis9, trans11-CLA, 1.3% is trans10, cis12-CLA, 11.6% is trans9 and trans 11-CLA.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> university of south of the Yangtze river
<120> linoleic acid isomerase and application thereof in conjugated linoleic acid production
<160> 16
<170> PatentIn version 3.3
<210> 1
<211> 330
<212> PRT
<213> Bifidobacterium breve (Bifidobacterium breve)
<400> 1
Met Leu Phe Gln Val Tyr Gly Asp Asn Ala Ile Tyr Gln Trp Ile Gly
1 5 10 15
Trp Ile Leu Val Phe Cys Cys Leu Ile Gly Ala Asn Glu Leu Ala Arg
20 25 30
Arg Thr Lys Thr Gly Gly Ile Val Ala Phe Leu Val Val Pro Ala Val
35 40 45
Leu Thr Val Tyr Phe Ile Thr Ile Tyr Thr Ala Ala Ala Met Gly Ala
50 55 60
Asp Trp Ala Leu Asn Asn Pro Thr Tyr Val His Met Thr Ser Trp Phe
65 70 75 80
His Tyr Ala Lys Leu Tyr Ala Ala Thr Ile Gly Cys Ile Gly Phe Met
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Ala Leu Lys Tyr Lys Trp Gly Ser Ile Gly Lys Ser His Trp Phe Lys
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Cys Phe Pro Phe Val Ile Val Ala Ile Asn Ile Leu Ile Ala Val Val
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Ser Asp Phe Glu Ser Ala Ile Arg Gly Trp Gly Thr Thr Trp Ile Ser
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Thr Glu Gly Val Thr Leu Tyr Gly Gly Trp His Asn Val Phe Asn Gly
145 150 155 160
Leu Ala Gly Ile Leu Asn Ile Phe Cys Met Thr Gly Trp Phe Gly Ile
165 170 175
Tyr Ala Ser Lys Lys Lys Asp Asp Met Leu Trp Pro Asp Met Thr Trp
180 185 190
Val Phe Ile Val Ala Tyr Asp Leu Trp Asn Phe Cys Tyr Thr Tyr Asn
195 200 205
Cys Leu Pro Thr His Ser Trp Tyr Cys Gly Leu Ala Leu Leu Leu Ala
210 215 220
Pro Thr Val Ala Asn Phe Phe Trp Asn Lys Gly Gly Trp Ile Gln Asn
225 230 235 240
Arg Ala Asn Thr Leu Ala Ile Trp Cys Met Phe Ala Gln Val Phe Pro
245 250 255
Met Phe Gln Asp Tyr Ser Val Phe Ser Thr Gln Ser Val Asn Asn Pro
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Asn Val Asn Leu Ala Val Ser Leu Ile Ala Leu Val Ala Asn Val Leu
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Ala Leu Gly Tyr Ile Leu Leu Arg Ala Lys Lys Gln Gly Ile Asn Pro
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Trp Thr Lys Glu Val Phe Lys Gly Thr Lys Asp Tyr Glu Gln Ala Ile
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Ala Arg Ala Asp Ala Ser Glu Leu Val Ala
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<210> 2
<211> 330
<212> PRT
<213> Bifidobacterium longum (Bifidobacterium longum)
<400> 2
Met Leu Phe Gln Val Tyr Gly Asp Asn Ala Ile Tyr Gln Trp Ile Gly
1 5 10 15
Trp Ile Leu Val Phe Cys Cys Leu Ile Gly Ala Asn Glu Leu Ala Arg
20 25 30
Arg Thr Lys Thr Gly Gly Val Ile Ala Phe Leu Val Ile Pro Ala Val
35 40 45
Leu Thr Val Tyr Phe Ile Thr Ile Tyr Thr Ala Ala Ala Met Gly Ala
50 55 60
Asp Trp Ala Leu Asn Asn Pro Thr Tyr Val His Met Thr Ser Trp Phe
65 70 75 80
His Tyr Ala Lys Leu Tyr Ala Ala Thr Ile Gly Cys Ile Gly Phe Met
85 90 95
Ala Leu Lys Tyr Lys Trp Gly Ser Ile Gly Lys Ser His Trp Phe Lys
100 105 110
Cys Phe Pro Phe Val Ile Val Ala Ile Asn Ile Leu Ile Ala Val Val
115 120 125
Ser Asp Phe Glu Ser Ala Ile Arg Gly Trp Gly Thr Thr Trp Ile Ser
130 135 140
Thr Glu Gly Val Thr Leu Tyr Gly Gly Trp His Asn Val Phe Asn Gly
145 150 155 160
Val Ala Gly Leu Leu Asn Ile Phe Cys Met Thr Gly Trp Phe Gly Ile
165 170 175
Tyr Ala Ser Lys Lys Lys Asp Asp Met Leu Trp Pro Asp Met Thr Trp
180 185 190
Val Phe Ile Val Ala Tyr Asp Leu Trp Asn Phe Cys Tyr Thr Tyr Asn
195 200 205
Cys Leu Pro Thr His Ala Trp Tyr Cys Gly Leu Ala Leu Leu Leu Ala
210 215 220
Pro Thr Val Ala Asn Phe Phe Trp Asn Lys Gly Gly Trp Ile Gln Asn
225 230 235 240
Arg Ala Asn Thr Leu Ala Ile Trp Cys Met Phe Ala Gln Val Phe Pro
245 250 255
Met Phe Gln Asp Tyr Ser Met Phe Ser Thr Gln Ser Val Asn Asn Pro
260 265 270
Asn Val Asn Leu Ala Val Ser Leu Ile Ala Leu Ala Ala Asn Val Leu
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Ala Leu Gly Tyr Ile Leu Leu Arg Ala Lys Lys Gln Gly Ile Asn Pro
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Trp Thr Lys Glu Val Phe Lys Gly Thr Lys Asp Tyr Glu Gln Ala Ile
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<210> 3
<211> 327
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<213> Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum)
<400> 3
Met Leu Phe Gln Val Tyr Gly Asp Thr Ala Ile Tyr Gln Trp Ile Gly
1 5 10 15
Trp Ile Leu Val Phe Cys Cys Leu Ile Gly Ala Asn Glu Leu Ala Arg
20 25 30
Arg Thr Lys Thr Gly Gly Val Ile Ala Phe Leu Ile Val Pro Ala Ile
35 40 45
Leu Thr Ile Tyr Phe Ile Thr Ile Tyr Val Ala Ala Ala Met Gly Ala
50 55 60
Glu Trp Ala Leu Ser Asn Pro Thr Tyr Val His Met Thr Ser Trp Phe
65 70 75 80
His Tyr Ala Lys Leu Tyr Ala Ala Thr Ala Gly Cys Ile Gly Phe Met
85 90 95
Ala Leu Lys Tyr Lys Trp Gly Lys Ile Gly Lys Ser Glu Trp Phe Lys
100 105 110
Cys Phe Pro Phe Val Ile Val Ala Ile Asn Ile Leu Ile Ala Val Ala
115 120 125
Ser Asp Phe Glu Ser Ala Ile Arg Ala Trp Gly Thr Thr Trp Val Ser
130 135 140
Thr Glu Gly Val Thr Leu Tyr Gly Gly Trp His Asn Val Phe Asn Gly
145 150 155 160
Val Ala Gly Leu Ile Asn Ile Ala Cys Met Thr Gly Trp Phe Gly Ile
165 170 175
Tyr Val Ser Lys Lys Lys Gln Asp Met Leu Trp Pro Asp Met Thr Trp
180 185 190
Val Phe Ile Val Ala Tyr Asp Ile Trp Asn Phe Cys Tyr Thr Tyr Asn
195 200 205
Cys Leu Pro Thr His Ser Trp Tyr Cys Gly Leu Ala Leu Leu Leu Ala
210 215 220
Pro Thr Val Ala Asn Phe Phe Trp Asn Lys Gly Gly Trp Ile Gln Asn
225 230 235 240
Arg Ala Asn Thr Leu Ala Ile Trp Cys Met Phe Ala Gln Val Phe Pro
245 250 255
Met Phe Gln Asp Glu Ser Lys Phe Ala Val Gln Ser Val Asn Asn Pro
260 265 270
Asn Val Asn Leu Thr Val Ser Ile Ile Ala Leu Val Ala Asn Val Leu
275 280 285
Ala Leu Gly Tyr Ile Met Tyr Arg Ala Lys Lys Gln His Val Asn Pro
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Trp Leu Gln Glu Val Phe Lys Gly Thr Arg Asp Tyr Glu Gln Ala Ile
305 310 315 320
Ala Arg Gln Glu Val Ala Ala
325
<210> 4
<211> 328
<212> PRT
<213> Bifidobacterium dentis (Bifidobacterium genus)
<400> 4
Met Leu Phe Gln Val Tyr Gly Asp Thr Ala Val Tyr Gln Trp Ile Gly
1 5 10 15
Trp Ile Leu Val Phe Cys Cys Leu Ile Gly Ala Asn Glu Leu Ala Arg
20 25 30
Arg Thr Lys Thr Gly Gly Ile Val Ala Phe Leu Val Val Pro Ala Ile
35 40 45
Leu Thr Val Tyr Phe Ile Thr Ile Tyr Val Ala Ala Ala Ala Gly Ala
50 55 60
Glu Trp Ala Leu Thr Asn Pro Thr Tyr Val His Met Thr Ser Trp Phe
65 70 75 80
His Tyr Ala Lys Leu Tyr Ala Ala Thr Ala Gly Cys Ile Gly Phe Met
85 90 95
Ala Leu Lys Tyr Lys Trp Gly Ala Ile Gly Lys Ser Glu Trp Phe Lys
100 105 110
Cys Phe Pro Phe Val Ile Val Ala Ile Asn Ile Leu Ile Ala Val Val
115 120 125
Ser Asp Phe Glu Ser Ala Ile Arg Ala Trp Gly Thr Thr Trp Val Ser
130 135 140
Thr Glu Gly Val Thr Leu Met Gly Gly Trp His Asn Val Phe Asn Gly
145 150 155 160
Val Ala Gly Leu Ile Asn Ile Ala Cys Met Thr Gly Trp Phe Gly Ile
165 170 175
Tyr Val Ser Lys Arg Lys Gln Asp Met Leu Trp Pro Asp Met Thr Trp
180 185 190
Val Phe Ile Val Ala Tyr Asp Leu Trp Asn Phe Cys Tyr Thr Tyr Asn
195 200 205
Cys Leu Pro Thr His Ser Trp Tyr Cys Gly Leu Ala Leu Leu Leu Ala
210 215 220
Pro Thr Val Ala Asn Phe Phe Trp Asn Lys Gly Gly Trp Ile Gln Asn
225 230 235 240
Arg Ala Asn Thr Leu Ala Ile Trp Cys Met Phe Ala Gln Val Phe Pro
245 250 255
Ala Phe Gln Asp Glu Ser Lys Phe Ala Val Gln Ser Val Asn Asn Pro
260 265 270
Asn Val Asn Leu Thr Val Ser Ile Ile Ala Leu Val Ala Asn Val Leu
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Ala Phe Gly Tyr Ile Met Tyr Arg Ala Arg Lys Gln His Val Asn Pro
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Trp Leu Gln Glu Val Phe Thr Gly Thr Lys Asp Phe Glu Gln Ala Met
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Ala Arg Arg Glu Asp Leu Ala Ala
325
<210> 5
<211> 993
<212> DNA
<213> Bifidobacterium breve (Bifidobacterium breve)
<400> 5
atgctgtttc aggtctacgg cgacaacgcc atctaccaat ggattggctg gatactcgtc 60
ttctgctgcc ttatcggcgc caatgaactg gctcgtcgca ccaaaaccgg cggcatcgtc 120
gccttcctcg tcgtcccggc tgtgctgacc gtctacttca tcaccatcta caccgccgcc 180
gcaatgggcg ccgactgggc actcaacaac ccgacctacg tgcacatgac cagctggttc 240
cactacgcca agctctacgc ggccaccatc ggctgcatcg gctttatggc cctcaaatac 300
aagtggggct ctatcggcaa atcccactgg ttcaagtgct tcccgttcgt gatcgtggcc 360
atcaacatcc tcatcgccgt ggtctctgac ttcgaatccg ccatccgcgg ctggggcacc 420
acctggatct ccactgaagg cgtgaccctc tacggtggct ggcacaacgt gttcaacggc 480
ttggccggca tcctcaatat cttctgcatg accggctggt tcggcatcta cgcctccaag 540
aagaaggacg acatgctctg gccggacatg acctgggtgt tcatcgtggc ctacgatctg 600
tggaacttct gctacaccta caattgcctg cccacccact cctggtactg cggccttgca 660
ctgctgctgg cgcccaccgt ggccaacttc ttctggaaca agggcggctg gatccagaat 720
cgcgccaata cattggccat ctggtgcatg ttcgcgcagg tattcccgat gttccaggac 780
tactccgtgt tctccaccca gtccgtgaac aacccgaacg tgaaccttgc ggtgtcccta 840
atcgcgctag tggccaacgt gttggcactc ggctacatcc tgctgcgcgc caagaagcag 900
ggcatcaacc cgtggaccaa ggaagtcttc aagggcacca aagactacga gcaggccatc 960
gctcgcgccg atgcatcgga gttggtggcg tag 993
<210> 6
<211> 993
<212> DNA
<213> Bifidobacterium longum (Bifidobacterium longum)
<400> 6
atgctgtttc aggtctacgg cgacaacgcc atctaccaat ggatcggatg gatactcgtc 60
ttctgctgtc ttatcggcgc caacgaactg gcccgccgca ccaaaaccgg tggcgttatc 120
gccttcctcg tcataccggc cgtgctgacc gtctacttca tcaccatcta cacggccgcc 180
gccatgggtg ccgactgggc cctcaacaac ccgacctacg tacacatgac cagctggttc 240
cactatgcca agctgtacgc ggccaccatc ggctgcatcg gtttcatggc cctcaaatac 300
aagtggggat ccatcggcaa atcgcactgg ttcaagtgct tcccgttcgt gatcgtggcc 360
atcaacatcc tcattgccgt agtgtccgac ttcgaatccg ccatccgcgg ctggggcacc 420
acgtggatct ccaccgaagg cgtgaccctg tacggtggct ggcacaacgt cttcaacggc 480
gtggccggcc tgctcaacat cttctgcatg accggctggt tcggcatcta cgcctccaag 540
aagaaggacg acatgctctg gccggacatg acctgggtgt tcatcgtggc ctacgacctg 600
tggaacttct gctacaccta caactgcctg cccacccacg cctggtattg cggcctggcg 660
ctgctgctgg cacccaccgt ggccaacttc ttctggaaca agggcggttg gattcagaac 720
cgcgccaaca cgctggccat ctggtgcatg ttcgcgcagg tgttcccgat gttccaggac 780
tattccatgt tctccaccca gtccgtgaac aatccgaatg tgaaccttgc agtctcgtta 840
atcgcgttgg cggccaatgt gctggcactt ggctacatcc tgctacgcgc caagaagcag 900
ggcatcaacc cgtggaccaa ggaagtgttc aaaggcacca aggattacga gcaggccatc 960
gctcgcgccg acgagtctga attggcggcc tag 993
<210> 7
<211> 984
<212> DNA
<213> Bifidobacterium pseudocatenulatum (Bifidobacterium pseudocatenulatum)
<400> 7
atgttgttcc aagtctatgg cgacaccgcc atataccagt ggatcggatg gatcctcgta 60
ttctgctgcc tgatcggcgc caatgagctg gcccgtcgca ccaagaccgg tggcgtgatc 120
gcgttcctga tcgtgccggc cattctgacc atctacttca tcaccattta cgtggccgcc 180
gcgatgggtg ccgaatgggc gctcagcaat ccgacctacg tgcatatgac cagctggttc 240
cactatgcca agctatatgc ggccaccgca ggctgcatcg gcttcatggc actcaaatac 300
aagtggggca agatcggcaa atccgaatgg ttcaagtgct tcccgttcgt gatcgtggcc 360
atcaacattc ttatcgccgt ggcctccgac ttcgaatcgg ccattcgcgc ttggggcacc 420
acatgggttt ccaccgaagg cgtgacgctg tatggcggct ggcacaatgt gttcaacggc 480
gttgccggcc tgatcaacat cgcctgcatg accggctggt tcggcattta cgtgtcaaag 540
aagaagcaag acatgctgtg gcctgacatg acttgggtat tcatcgtcgc atacgatatt 600
tggaacttct gctacaccta caactgcctg ccgacccact cctggtattg cggcctcgcg 660
ctgctgctcg ccccgaccgt ggcgaacttc ttctggaaca agggcggctg gatccagaac 720
cgcgccaaca cgctcgccat ctggtgcatg ttcgcgcagg tgttccccat gttccaggat 780
gagtccaagt tcgccgtgca gtcggtgaac aatccgaacg tgaacctgac cgtgtcgatc 840
atcgcgctcg tggccaacgt gctcgcactc ggctacatca tgtaccgcgc gaagaagcag 900
cacgtcaacc cgtggctgca ggaagtcttc aagggcaccc gcgactacga gcaggccatc 960
gcccgccagg aagtcgccgc ctga 984
<210> 8
<211> 986
<212> DNA
<213> Bifidobacterium dentis (Bifidobacterium genus)
<400> 8
atgttgttcc aagtctatgg cgacaccgcc gtatatcagt ggatcggctg gatccttgtc 60
ttctgctgcc tgatcggcgc caatgagctg gcccgccgca ccaagaccgg cggtatcgtg 120
gcgttcctgg tcgttcctgc gatcctgacc gtctatttca tcaccatcta tgtagccgct 180
gccgccggcg ccgaatgggc gctgaccaac ccgacctacg tgcacatgac cagctggttc 240
cattatgcga aactgtacgc agcgaccgcg ggatgcatcg gcttcatggc actcaagtac 300
aagtggggcg ccatcggcaa atccgaatgg ttcaaatgct tcccgttcgt aatcgtggcc 360
atcaacatcc tcatcgccgt ggtttccgac ttcgaatccg cgatccgcgc atggggcacc 420
acctgggtct ccaccgaagg cgtgacgctc atgggcggct ggcacaacgt gttcaacggc 480
gtggcgggcc tcatcaacat cgcctgcatg accggatggt tcggcatcta cgtgtcgaag 540
aggaagcagg acatgctctg gcccgacatg acgtgggtgt tcatcgtagc ctacgacctg 600
tggaacttct gctacaccta caactgcctg cccacccact cgtggtactg cggtctggcg 660
ctgctgcttg caccgaccgt cgccaacttc ttctggaaca agggcggctg gattcagaac 720
cgcgccaaca cactcgccat ctggtgcatg ttcgcgcagg tgttccctgc cttccaggac 780
gagtccaagt tcgccgtgca gtcggtgaac aacccgaacg tgaacctgac cgtgtcgatc 840
atcgcactcg tggcgaacgt gctcgcattc ggctatatca tgtaccgtgc caggaagcag 900
cacgtgaacc cgtggctgca ggaggtgttc acggcaccaa ggactttgag caggccatgg 960
cccgccgcga agatctggcg gcctga 986
<210> 9
<211> 29
<212> DNA
<213> Artificial sequence
<400> 9
aagcctatgc tgtttcaggt ctacggcga 29
<210> 10
<211> 25
<212> DNA
<213> Artificial sequence
<400> 10
catatgctac gccaccaact ccgat 25
<210> 11
<211> 25
<212> DNA
<213> Artificial sequence
<400> 11
aagcctatgc tgtttcaggt ctacg 25
<210> 12
<211> 25
<212> DNA
<213> Artificial sequence
<400> 12
catatgctag gccgccaatt cagac 25
<210> 13
<211> 25
<212> DNA
<213> Artificial sequence
<400> 13
aagcctatgt tgttccaagt ctatg 25
<210> 14
<211> 25
<212> DNA
<213> Artificial sequence
<400> 14
catatgtcag gcggcgactt cctgg 25
<210> 15
<211> 25
<212> DNA
<213> Artificial sequence
<400> 15
aagcctatgt tgttccaagt ctatg 25
<210> 16
<211> 25
<212> DNA
<213> Artificial sequence
<400> 16
catatgtcag gccgccagat cttcg 25
Claims (10)
1. A linoleic acid isomerase enzyme, wherein said linoleic acid isomerase enzyme is:
(a) a protein consisting of an amino acid sequence shown by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4; or,
(b) and (b) the protein which is derived from the protein (a) and has linoleate isomerase activity, wherein the amino acid sequence in the protein (a) is subjected to substitution, deletion or addition of one or more amino acids.
2. A gene encoding the linoleate isomerase enzyme of claim 1.
3. The gene as claimed in claim 2, characterized in that the nucleotide sequence of the gene is shown as SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.
4. A recombinant plasmid carrying the gene of claim 2 or 3.
5. A host cell carrying the gene of claim 2 or 3 or the recombinant plasmid of claim 4.
6. The host cell of claim 5, wherein the host cell is E.coli.
7. Use of the linoleate isomerase according to claim 1 or the gene according to claim 2 or 3 or the recombinant plasmid according to claim 4 or the host cell according to claim 5 or 6 for the production of conjugated linoleic acid.
8. A method for producing conjugated linoleic acid, which comprises inoculating the host cell of claim 5 or 6 into a culture medium, and culturing the host cell to OD at 35-40 ℃ and 150-250 rpm6000.4-0.6 to obtain a culture solution A; adding IPTG with the final concentration of 0.01-1.0 mM into the culture solution A, and carrying out induced culture for 12-16 h under the conditions that the temperature is 15-20 ℃ and the rotating speed is 150-250 rpm to obtain a culture solution B; centrifuging the culture solution B, and collecting wet thalli; adding wet bacteria into a reaction system containing linoleic acid by taking the linoleic acid as a substrate, and reacting at the temperature of 35-40 ℃ and the rotating speed of 150-250 rpm to obtain a reaction solution rich in conjugated linoleic acid; extracting the reaction liquid rich in the conjugated linoleic acid to obtain the conjugated linoleic acid.
9. The method of claim 8, wherein the conjugated linoleic acid is cis9, trans11-CLA and/or trans9, trans 11-CLA.
10. A method for producing the linoleic acid isomerase of claim 1, wherein the method comprises the steps of adding the host cell of claim 5 or 6 into a culture medium, culturing at the temperature of 35-40 ℃ and the rotation speed of 150-250 rpm to obtain a host cell rich in linoleic acid isomerase, and extracting the host cell rich in linoleic acid isomerase to obtain the linoleic acid isomerase.
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| CN201911011728.3A CN112695024B (en) | 2019-10-23 | 2019-10-23 | Linoleic acid isomerase and application thereof in conjugated linoleic acid production |
| PCT/CN2019/121822 WO2021077543A1 (en) | 2019-10-23 | 2019-11-29 | Linoleate isomerase and use thereof in production of conjugate linoleic acid |
| US17/497,963 US12123037B2 (en) | 2019-10-23 | 2021-10-10 | Linoleic acid isomerase and its application in production of conjugated linoleic acid |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103087935A (en) * | 2013-01-16 | 2013-05-08 | 天津科技大学 | Fermentation method of conjugated linoleic acid and strain used in method |
| CN105925514A (en) * | 2016-07-12 | 2016-09-07 | 江南大学 | Bifidobacterium breve and application thereof in preparing conjugated linoleic acid or conjugated linolenic acid |
| CN106148230A (en) * | 2016-07-12 | 2016-11-23 | 江南大学 | One strain vacation chainlet bacillus bifidus and prepare the application of conjugated linoleic acid or conjugate linolenic acid |
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2019
- 2019-10-23 CN CN201911011728.3A patent/CN112695024B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103087935A (en) * | 2013-01-16 | 2013-05-08 | 天津科技大学 | Fermentation method of conjugated linoleic acid and strain used in method |
| CN105925514A (en) * | 2016-07-12 | 2016-09-07 | 江南大学 | Bifidobacterium breve and application thereof in preparing conjugated linoleic acid or conjugated linolenic acid |
| CN106148230A (en) * | 2016-07-12 | 2016-11-23 | 江南大学 | One strain vacation chainlet bacillus bifidus and prepare the application of conjugated linoleic acid or conjugate linolenic acid |
Non-Patent Citations (3)
| Title |
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| 匿名: "CP034192.1", 《GENBANK》 * |
| 匿名: "UniProtKB - S2ZIT7", 《UNIPROTKB》 * |
| 匿名: "WP_003827883.1", 《GENBANK》 * |
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